iniae HD-1 BI-D1870 using rabbit anti-MtsA antibodies (Figure 5A). MtsA was detected in the particulate fraction of the cells when the cellular fractions were prepared by centrifugation of the crude cell lysate (the first treatment). MtsA was found to be associated with the protoplast and cell wall extracts when the cellular fractions were prepared by protoplast

formation. After separation of the protoplasts, MtsA was detected in the particulate fraction (the second treatment). Figure 5 Detection of the subcellular localization of MtsA in S. iniae HD-1 by western blotting. (A) The cellular fractions of S. iniae HD-1 and rabbit anti-MtsA antibodies were used for the western-blot assay. Lane 1, S. iniae HD-1 selleck chemicals llc lysate; lane 2, soluble fraction of cells; lane 3, particulate fraction of cells; lane 4, cell wall extracts; lane 5, protoplast; lane 6, particulate fraction of protoplasts; and lane 7, soluble fraction of protoplasts. (B) Surface exposure of MtsA. Cells (lanes 1 and 2), cell wall extracts (lanes 3 and 4), and protoplasts (lanes 5 and 6) of S. iniae HD-1 were treated with proteinase K and analyzed by western blotting. Lanes 1, 3 and 5 show the untreated control, while lanes 2, 4 and 6 show samples treated with proteinase K for 1 h. To detect surface exposure of MtsA, cells of S. iniae

HD-1 cells were harvested, washed, centrifuged, and resuspended in PBS. The cells were subjected to proteinase K (5 μg ml-1) treatment with gentle agitation Resveratrol at room temperature for 1 h, and the cells were collected. Western blotting showed that peptide MK5108 price fragments in the cells can be detected after 1 h incubation with proteinase K. However, when the cell wall

extracts and protoplasts were used in the experiment, it were completely hydrolyzed and no peptide fragments were detected (Figure 5B). Together, this result indicated that MtsA is not exposure on surface, but is on the outside of the cytoplasmic membrane and is buried inside the cell wall. MtsA had heme-binding activity To examine whether heme is the chromophore associated with MtsA, the pyridine hemochrome assay was performed [28]. The UV-visible absorption spectrum of purified MtsA exhibited peaks at 275, 420, 525, and 560 nm, which were identical to those obtained from purified KatG, a well-known heme-containing protein with spectral peaks at 418, 524, and 556 nm. The molar ratio of associated heme to purified MtsA was 0.806 (Figure 6), this value is consistent with the hypothesis that one protein molecule is associated with one heme molecule. Figure 6 Detection of the heme-binding activity of purified MtsA by the pyridine hemochrome assay. (A) UV-visible absorption spectrum of 20 μM purified MtsA (■ line) in 50 mM Tris-HCl (pH 8.0). (B) UV-visible absorption spectrum of 20 μM purified KatG (Δ line) in 50 mM Tris-HCl (pH 8.0).